In natural
habitats microorganisms usually grow in complex, mixed populations containing
several species. This presents a problem for the microbiologist because a
single type of microorganism cannot be studied adequately in a mixed culture. One
needs a “pure culture”- that's a population of cells arising from a single cell
to characterize an individual species. The development of pure culture
techniques by the German bacteriologist Robert Koch transformed microbiology.
Within about 20 years after the development of pure culture techniques, most
pathogens responsible for major human bacterial diseases had been isolated.
A variety of techniques have been developed whereby isolation into pure culture can be accomplished. Each technique has certain advantages and limitations, and there is no one method that can be used for all bacteria. Most commonly used pure culture techniques include streak plate method, spread plate method and pour plate method. Streak plate is considered only as a qualitative method. Unlike the streak plate technique, the pour plate and spread plate techniques may be performed in a quantitative manner to determine the number of bacteria present in a specimen.
The streak culture
or surface plating method is routinely employed for the isolation of bacteria
in pure culture from clinical specimens. A platinum loop is charged with the
specimen to be cultured. Owing to the
high cost of platinum, loops for routine work are made of nichrome resistance
wires. One loopful of specimen is transferred onto the surface of a well dried
agar plate on which it is spread over a small area at the periphery. It is
called the primary inoculum. The primary inoculum is then distributed over the
plate by streaking it with the loop in a series of parallel lines in different
segments of the plate. The loop should be flamed and cooled between the
different sets of streaks. At some point in the process, single cells dropped
from the loop as it is rubbed along the agar surface would develop into
separate colonies on incubation. Growth may be confluent at the site of
original inoculation but become progressively thin and well separated colonies
are obtained over the final series of streaks. Common streaking methods include T- streaking, Zig-zag streaking, Continuous streaking and Quadrant streaking (Figure 1).
Fig
1. Different streaking patterns
A) The pour plate technique
A pour plate can
yield isolated colonies and extensively used with the bacteria and fungi. The original
sample is diluted several times to reduce the microbial population sufficiently
to obtain separate colonies when plating (Figure 2). Serial dilution is the
method commonly used to dilute the original sample. For serial dilution, a
series of tubes containing a definite volume of sterile liquid, usually water
or physiological saline is prepared. Suppose we prepare a series of tubes
containing 9 ml of sterile distilled water. For carrying out serial dilution of
the original sample we can add 1 ml of the original sample into the first tube
which contains 9 ml of sterilized distilled water. Now the final volume of the
first tube becomes 10. So the dilution of the first tube is 1/10 which can be
also written as 10-1. To continue the serial
dilution, from the first tube we can add
1 ml to the second tube. This is continued in the following tubes also till we
reach the final tube. From the final tube, remove 1ml of the media, so that the
volume will be 9 ml in all the tubes. From these diluted tubes small volumes
of several diluted samples are mixed with liquid Agar that has been cooled to
about 45 degrees celsius. The mixtures are then poured immediately into sterile
culture dishes. Most bacteria and fungi are not killed by a brief exposure to
the warm Agar. After the agar has hardened, each cell is fixed in place and
forms individual colony. Plates
containing between 30 to 300 colonies are counted. The total number of colonies
equals the number of viable microorganisms in the diluted sample. Colonies growing
on the surface can be used to inoculate fresh medium and to prepare pure
cultures. This is considered as a quantitative as well as qualitative method.
This is a preferred quantitative method for urine cultures. Surface colonies
formed by pour culture technique are circular and subsurface colonies could be
lenticular or lens shaped.
Disadvantages
1.
Some of the organisms are
trapped beneath the surface of the medium during pour plate technique and
therefore both surface and the subsurface colonies develop. The subsurface colonies can be transferred to
fresh media only by first digging them out of the agar with a sterile
instrument. So, there is more chance for contamination.
2. The organisms being isolated must be able to withstand temporary exposure to 45 degree celsius, temperature of the liquid agar medium. So, this method would be unsuitable for isolating psychrophilic bacteria. (Psychrophiles are the bacteria that grow well at 200 C degree and have an optimum growth temperature of 150 C or lower and maximum around 200 C)
Figure- 2. The
pour plate technique- The original sample is diluted several times to thin out
the population sufficiently. The most diluted samples are then mixed with warm
agar and poured into petri dishes. Isolated cells grow into colonies and can be
used to establish pure cultures.
CFU/ml (Colony
Forming Unit/ml)= (No: of colonies X Dilution factor) / Volume of sample plated
Dilution factor =
Reciprocal of dilution or Final volume/ sample volume
(If 1 ml sample is
added to 9 ml, its dilution = 1/10= 10 -1; Dilution factor is 10)
C) Spread plate technique
The spread plate
is an easy, direct way of achieving pure cultures. This method is also
considered as a quantitative as well as qualitative method. In this method also, serial dilution has to
be conducted initially. The original culture is diluted in a series of tubes
containing sterile liquid, usually water or physiological saline. After that, a
small volume of dilute microbial mixture containing around 30- 300 cells is
transferred to the center of a dry Agar plate. Then it is spread evenly over
the surface of agar media with a sterile
glass rod or the L-rode. The dispersed cells develop into isolated
colonies. The number of colonies developed on the plates would be equal to the
number of viable organisms, as each viable cell develops into a colony on
incubation. In contrast to the pour plate technique, only surface colonies
develop in the pour plate technique. Moreover the organisms are not required to
withstand the temperature of liquid agar as in the case of pour plate
technique.
Preparation of
spread plate
●
Pipette a small volume (0.01ml) of sample on
to the center of an agar medium plate
●
Dip the
glass spreader into a beaker of ethanol
●
Briefly flame the ethanol soaked spreader and
allow it to cool
●
Spread the sample evenly over the agar surface
with the sterilized spreader and incubate (Fig 3)